The present invention relates to regulators of MMP-9 and, more particularly, to regulators targeted towards the OG domain thereof.
The physiological and pathological roles of matrix metalloproteinases (MMPs) are versatile. Members of the MMP family have been implicated in numerous aspects of the migration of inflammatory and cancer cells through connective tissues, not only by catabolizing extracellular matrix (ECM) components but also by processing various soluble mediators, promoting many disease states. Although all MMPs share similar catalytic sites, marked differences are observed in their substrate specificity, at least in part due to the presence of additional substrate binding sites in non-catalytic protein domains. As a consequence, different MMPs have different biological functions. MMP-9, also known as gelatinase B, is a prototypical target in inflammatory diseases, because of its tissue-damaging roles and inflammation-promoting processing of soluble proteins, including protease inhibitors, chemokines and cytokines.
In contrast, MMP-2 or gelatinase A has mainly anti-inflammatory and homeostatic functions, presumably by the inactivation of inflammatory chemokines and by regulating connective tissue turn-over. This implies that selective inhibitors, discriminating between these highly similar enzymes, are crucial for efficient anti-inflammatory therapy without side-effects. In this perspective, other non-catalytic parts of the enzyme, differentiating MMP-2 and MMP-9, may be targeted to generate selective inhibitors.
Interestingly, the main structural difference between MMP-9 and MMP-2 is the presence of an extensively O-glycosylated (OG) domain in MMP-9 [Opdenakker, G., et al (2001), Trends Immunol. 22, 571-579; Van den Steen, P. E., et al (2006) J Biol Chem. 281, 18626-18637]. Other domains in MMP-9 are also found in MMP-2 and include a pro-peptide domain responsible for maintaining latency, a catalytic domain in which three fibronectin repeats are inserted, and a C-terminal domain also known as the hemopexin-like domain which constitutes an exosite for binding of the endogenous MMP-9 and MMP-2 inhibitor, tissue inhibitor of metalloproteinase 1 (TIMP-1). Despite its great importance in many disease states and in contrast to MMP-2, the available structural information about MMP-9 is limited to its two terminal domains, rather than the full length enzyme. The X-ray structure of the N-terminal part [Elkins et al, 2002, Acta Crystallogr D Biol Crystallogr 58, 1182-1192], containing the pro-catalytic domain shows that it possesses a matrixin fold. The C-terminal hemopexin-like domain consists of a four-bladed β-propeller structure with pseudo-four-fold symmetry [Cha et al, 2002, J Mol Biol 320, 1065-1079]. FIG. 1A presents the crystal structures of the pro-catalytic and the hemopexin-like domains of pro-MMP-9. The domains are connected by a dotted line representing the 64 amino acid-long linker (containing 22 proline residues, 6 glycine residues and approximately 12-14 O-linked glycans [Van den Steen et al., 2001, Biochim Biophys Acta 1528, 61-73]. Importantly, the linker domain of pro-MMP-9 is 2-3 times longer than linker regions of collagenases, stromelysins and gelatinase A, of the MMP family, for which typical linker lengths span a range of only 21-27 amino acid residues.
Crystallization of the linker domain in pro-MMP-9 separately or together with other protein domains has proven difficult. The lack of a large side chain in the case of glycine and the presence of a built-in bend in the case of proline interfere with the formation of secondary structure and often result in loops or unstructured regions. In addition, the presence of clustered serines and threonines as attachment points for O-glycans might yield steric effects that could hinder crystallographic packing. This domain has also been termed the collagen V-like domain, due to its sequence similarity to collagen V and has recently been renamed O-glycosylated (OG) domain. The OG domain is active in the orientation of the hemopexin domains to enable exosite interactions. However, nothing is known of the influence of the OG domain on the overall 3D structure of MMP-9 and its biophysical nature.
U.S. Patent No. 20040175817 teaches identification of MMP-9 modulators based on the crystal structure of its catalytic subunit. However, since MMPs in general share a high sequence homology in their catalytic sites, modulators designed to target the catalytic site will not be selective towards MMP-9.